The long-term objectives of this project are to determine whether PT activity from B. pertussis infection exacerbates a subsequent influenza virus infection and to determine the mechanisms involved. B.pertussis causes a severe respiratory disease that is frequently complicated by respiratory viral infections that can result in death. We know from our own research that PT is important during the early stages of B. pertussis infection by targeting alveolar macrophages, altering their production of inflammatory cytokines PT and delaying the recruitment of neutrophils to the airways. Presumably these and other immunosuppressive effects inhibit innate immune responses to B. pertussis and other pathogens, which can increase the risk of secondary infections. Based on our preliminary observations we hypothesize that: (1) B. pertussis through PT activity can exacerbate a subsequent virus infection and (2) that PT does so by suppressing the type I IFNs of the antiviral response. To achieve our gaols and test our hypotheses we will examine viral replication of influenza A/PR/8/34, a mouse adapted strain, in mice infected with B. pertussis or treated with PT. We will determine if PT enhances viral titers, exacerbates disease and increases mortality. We will also determine if PT suppresses the antiviral response by antagonizing induction, signaling and gene activation of type I interferons. We will assay IFN bioactivty in lung epithelial and alveolar macrophage cell lines by IFN bioassay. Type I IFN induction in mice will be assayed by ELISA, IFN Bioassay and RT-PCR. Signaling through the IFN pathway will be tested by assaying the activation of key transcription factors activated during infection(IRF-3, IRF-7, STAT1). We will also test whether PT can rescue the growth of a mutant virus,influenza A ?NSI, that can not antagonize type I IFN reponses. Lastly we will examine gene expression for five IFN stimulated genes: protein kinase PKR, 2 ,5 -Oligoadenylate Synthetase, RNaseL, Adenosine deaminase (ADAR1) and MX protien GTPase by RT-PCR. Numerous studies indicate that patients infected with B. pertussis are often co-infected with one or more viruses, such as influenza virus, respiratory syncytial virus (RSV), and adenovirus. Therefore understanding the underlying mechanisms of how B. pertussis infection manipulates the immune response will contribute to our overall knowledge about the pathogenesis of B. pertussis but more importantly lead to the development of new and improved diagnostics and treatment for coinfections.